Biosample Manipulation Apparatus

ABSTRACT

A biosample manipulation method according to the invention includes the steps of: providing a scanning probe microscope ( 3 ) with a cantilever ( 9 ) including a beam portion ( 15 ) with its base end supported in a cantilever manner, a probe ( 17 ) formed to protrude from the beam portion ( 15 ) and having a hollow ( 16 ) in itself, a fine hole ( 21 ) formed to penetrate from the hollow ( 16 ) to an outside of the probe ( 17 ) through the probe ( 17 ), and a first electrode ( 20 ) provided in the hollow ( 16 ): filling fluid ( 19 ) including a physiologically active substance or a substance for exciting a chemical reaction and having conductivity in the hollow ( 16 ) in the cantilever ( 9 ); placing a biosample ( 2 ) on a second electrode ( 12 ) formed of a conductive thin film: bringing the probe ( 17 ) of the cantilever ( 9 ) close to the biosample ( 2 ); applying a pulse voltage between the first electrode ( 20 ) and the second electrode ( 12 ) to thereby allow the fluid ( 19 ) to flow out of the fine hole ( 21 ) and inject the fluid ( 19 ) into the biosample ( 2 ); and observing change in a surface profile of the biosample ( 2 ) through the scanning probe microscope.

TECHNICAL FIELD

The present invention relates to a biosample manipulation apparatus forinjecting or dropping a medicinal solution including a gene, protein, anenzyme, or the like into or on a biosample such as a cell andparticularly to a biosample manipulation apparatus using a cantilever.

BACKGROUND TECHNIQUE

Conventionally, in a field of biological research, pathologic research,or the like, a gene or the like is injected into and expressed in acell. Here, as a means for injecting the gene or the like into abiosample such as a cell, a method has been conventionally proposedusing a cantilever provided to a scanning probe microscope (see PatentDocument 1). FIG. 8 is a drawing showing an example of a prior-artbiosample manipulation apparatus. As shown in the drawing, the biosamplemanipulation apparatus 80 includes a cantilever 82 provided to ascanning probe microscope (not shown) and protruding a probe 81 in asharp-pointed shape from a tip end portion of the cantilever 82 and aneedle-like object 83 attached to a tip end of the cantilever 82 andformed of a carbon nanotube or the like. In using this biosamplemanipulation apparatus 80, a gene or the like is fixed to a tip end ofthe needle-like object 83, the cantilever 82 is caused to scan to insertthe needle-like object 83 into a cell 84 to thereby introduce the geneor the like into the cell 84, and this state is retained to express thegene.

On the other hand, the present applicant has conventionally proposed amicro-machining apparatus using a cantilever (see Patent Document 2).This micro-machining apparatus includes a cantilever portion supportedin a cantilever manner and a protruding portion formed to protrude froma tip end of the cantilever portion. A hollow is formed in theprotruding portion and a fine hole leading outside the protrudingportion from the hollow is formed to penetrate the protruding portion.Here, in the hollow in the protruding portion, fluid such as indium,gallium, an alloy of indium and gallium, or the like is filled. In usingthe micro-machining apparatus, by application of a pulse voltage to thecantilever, the fluid flows outside through the fine hole to form anultrafine dot or a ultrafine line of In, Ga, or the like on a surface ofa sample. In this way, a semiconductor light emitting device or the likefor high-speed optical communication is produced.

However, in the prior-art biosample manipulation apparatus 80, it isnecessary to fix the gene or the like to be introduced into the cell 84to the tip end of the needle-like object 83 and therefore there is aproblem that a medicinal liquid in which a gene, a chemical, or the likeis dissolved or fluid such as a reactive gas cannot be injected ordropped into the cell 84.

Moreover, since the needle-like object 83 having a diameter of about 10nm to 30 nm is inserted into the cell, a hole 86 greater than thediameter of the needle-like object 83 is formed in a cell membrane 85 orthe needle-like object 83 may damage a cell nucleus 87 in some cases. Asa result, there are also problems that the cell 84 may suffer greatdamage and die or it takes the cell 84 a long time to repair the hole 86or damage by its self-repairing function.

Patent Document 1: Japanese Patent Application Laid-open No. 2003-325161

Patent Document 2: Japanese Patent Application Laid-open No. 2004-34277

DISCLOSURE OF THE INVENTION

The present invention has been made with the above problems in view andit is an object of the invention to provide a means for injecting ordropping fluid such as medicinal liquid in which a gene or the like isdissolved into a biosample while minimizing damage caused to thebiosample at this time.

According to the invention, a biosample manipulation apparatus forinjecting a physiologically active substance or a substance for excitinga chemical reaction into a biosample includes: a beam portion with itsbase end supported in a cantilever manner; a probe formed to protrudefrom a tip end of the beam portion and having a hollow in itself a finehole formed to penetrate from the hollow to an outside of the probethrough the probe; fluid filled in the hollow; and a fluid injectingmeans for allowing the fluid to flow out of the fine hole and injectingthe fluid into the biosample. According to the invention, the fluidinjecting means can allow a minute amount of fluid filled in the hollowin the probe to flow out of the fine hole and inject the fluid into thebiosample.

Moreover, according to the invention, the fluid injecting meansincludes: a first electrode provided in contact with the fluid; a secondelectrode provided in contact with the biosample; and a pulsed powersupply for applying a pulse voltage between the first electrode and thesecond electrode. According to the invention, by applying the pulsevoltage to the fluid, binding force acting between respective particlesforming the fluid is cut off and the fluid that has lost cohesion flowsout of the fine hole. By applying the pulse voltage to the biosample, ahole is formed in a surface of the biosample due to a transitorydielectric breakdown and the fluid passes through the hole and is takeninto the biosample due to electrophoresis. Since the hole is extremelyminute and closes up soon due to a self-repairing function of thebiosample, the biosample does not suffer much damage. In this way, it ispossible to inject the fluid into the biosample while minimizing thedamage caused to the biosample.

Furthermore, according to the invention, the biosample manipulationapparatus for dropping a physiologically active substance or a substancefor exciting a chemical reaction on a biosample includes: the beamportion with its base end supported in the cantilever manner; the probeformed to protrude from a tip end of the beam portion and having ahollow in itself; a fine hole formed to penetrate from the hollow to anoutside of the probe through the probe; fluid filled in the hollow; anda fluid outflow means for allowing the fluid to flow out of the finehole. According to the invention, the fluid outflow means can allow aminute amount of fluid filled in the hollow in the probe to flow out ofthe fine hole and drop the fluid on the biosample.

Moreover, according to the invention, the fluid outflow means includes apair of electrodes provided in contact with the fluid and the pulsedpower supply for applying a pulse voltage between the respectiveelectrodes. According to the invention, by applying the pulse voltage tothe fluid, binding force acting between respective particles forming thefluid is cut off and the fluid flows out of the fine hole.

Furthermore, according to the invention, the beam portion, the probe,and the fine hole constitute a probe of a scanning probe microscope.According to the invention, after manipulation of the biosample, it ispossible to check if a medicinal liquid or the like has been injectedinto or dropped on the biosample reliably and a degree of breakage ofthe biosample due to application of the pulse voltage.

Moreover, the invention comprises a manipulation observing means forobserving manipulation of the biosample and change of the biosampleafter the manipulation. According to the invention, it is possible toeasily select a biosample to be manipulated from a plurality of arrangedbiosamples and to manipulate the biosample more accurately whileobserving through the manipulation observing means. It is also possibleto observe the inner change occurring in the biosample after themanipulation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic illustration of a biosample manipulationapparatus 1, 30, 40, 50, or 60 according to an embodiment of theinvention.

FIG. 2 is a partially enlarged sectional view of a vicinity of a cell 2in FIG. 1.

FIG. 3 is a schematic perspective view of a structure of a cantilever 9according to a first embodiment.

FIG. 4 is a schematic perspective view of a structure of a cantilever 31according to a second embodiment.

FIG. 5 is a schematic perspective view of a structure of a cantilever 41according to a third embodiment.

FIG. 6 is a schematic perspective view of a structure of a cantilever 51according to a fourth embodiment.

FIG. 7 is a schematic perspective view of a structure of a cantilever 61according to a fifth embodiment.

FIG. 8 is a schematic side view of a prior-art biosample manipulationapparatus 80.

FIG. 9 is an attached sheet for explaining reference numerals used inthe respective drawings.

BEST MODES FOR CARRYING OUT THE INVENTION

A first embodiment of the present invention will be described below withreference to the drawings. FIG. 1 is a diagrammatic illustration of abiosample manipulation apparatus 1 according to the present embodiment.As shown in the drawing, the biosample manipulation apparatus 1 includesan atomic force microscope (hereafter referred to as “AFM”) 3 formanipulating a cell (biosample) 2 as an object of manipulation and forobserving change of a surface of the cell 2, a pulsed power supply 4 forapplying a pulse voltage to the AFM 3, a control portion 5 forcontrolling operations of the AFM3 and the pulsed power supply 4, acomputer 6 which is an input/output portion of the control portion 5,and an optical microscope (manipulation observing means) 7 for observingthe cell manipulation and inner changes of the cell 2 after themanipulation. Although the cell 2 is taken as an example of thebiosample in description of the embodiment, it is also possible to use apiece of tissue of a living thing or a biopolymer such as a protein andan enzyme besides the cell.

The AFM3 is a type of a scanning probe microscope and is for observing asurface profile of the cell 2 and manipulating the cell 2. As shown inFIG. 1, the AFM3 includes a scanning stage 8 on which the cell 2 isplaced and a cantilever 9 for scanning a surface of the cell 2 on thescanning stage 8.

FIG. 2 is a partially enlarged sectional view of a vicinity of a cell 2in FIG. 1. As shown in FIGS. 1 and 2, the scanning stage 8 includes astage main body 10 in which a piezoelectric device actuator (not shown)is mounted, a glass plate 11 disposed on an uppermost portion of thestage main body 10, and a transparent conductive thin film (secondelectrode) 12 stuck on an upper face of the glass plate 11. In a statein which the cell 2 is placed on the conductive thin film 12, it ispossible to move the cell 2 in directions of X, Y, and Z axes byapplying a voltage to the piezoelectric device actuator to expand andcontract the scanning stage 8. As shown in FIG. 1, a cavity 13vertically penetrating the stage main body 10 is formed in the stagemain body 10 and the optical microscope 7 is disposed below the cavity13. In this way, through the glass plate 11 and the conductive thin film12, it is possible to observe a manner of manipulation of the cell 2,changes in the cell 2, and the like through the optical microscope 7.

FIG. 3 is a schematic perspective view of a structure of a cantilever 9.As shown in FIGS. 1 to 3, the cantilever 9 includes a beam portion 15with its base end supported in a cantilever manner by a holder 14, aprobe 17 formed to protrude from a tip end of the beam portion 15 andhaving a hollow 16, a mask 18 applied to an inner wall face of the probe17, medicinal liquid 19 filled in the hollow 16, a conductive thin film(first electrode) 20 provided to extend from a surface of the beamportion 15 to the inner wall face of the probe 17, and a fine hole 21formed to penetrate the probe 17 from the hollow 16 to an outside of theprobe 17.

The probe 17 comes extremely close to the surface of the cell 2 todetect a surface profile of the cell 2. The probe 17 is in a shape of aquadrangular pyramid and the hollow 16 is formed to extend from a bottomface side toward a vertex side of the quadrangular pyramid in a shape ofa quadrangular pyramid as shown in FIG. 3. The mask 18 lowers surfacetension of the medicinal liquid 19 to thereby make the medicinal liquid19 liable to flow outside through the fine hole 21. In the embodiment,the thin film is formed by applying gold to the inner wall face of theprobe 17. In the invention, the mask 18 is an arbitrary component andingredients, a film thickness, and the like of the mask 18 can beproperly changed in design. The medicinal liquid 19 is prepared bydissolving a nucleic acid substance such as DNA or a protein substancesuch as an antigen, an enzyme, and a hormone (hereafter they will bereferred to as “physiologically active substances”) in a buffer solutionand has conductivity. Since an opening diameter of the fine hole 21 isextremely minute and respective particles forming the medicinal liquid19 are bound together by action of atomic force or the like, thismedicinal liquid 19 is retained in the probe 17 without flowing outsidethe probe 17 through the fine hole 21. The medicinal liquid 19 may beone exciting a chemical reaction when a minute amount of the liquid isdropped on the cell 2 besides the liquid in the embodiment. The finehole 21 functions as an outlet for allowing the medicinal liquid 19filled in the hollow 16 to flow outside the probe 17. The fine hole 21is an ultrafine hole having a circular cross section and an openingdiameter of 20 to 500 nm. The fine hole 21 is formed from a vertex ofthe hollow 16 toward a vertex of the probe 17 as shown in FIG. 3.Therefore, the vertex of the probe 17 is chipped and the tip end of theprobe 17 is formed of a section of the fine hole 21. By bringing thissection extremely close to the surface of the cell 2, the surfaceprofile is scanned. By changing a size of the cross section of the finehole 21, it is possible to adjust an amount of outflow of the medicinalliquid 19. The conductive thin film 20 is for applying the pulse voltageto the medicinal liquid 19 and the cell 2. This conductive thin film 20is stuck on an upper face of the beam portion 15 in a vertical directionof the beam portion 15 and a tip end portion of the film 20 is bent downand stuck along the inner wall face of the probe 17 as shown in FIGS. 2and 3. Therefore, when the medicinal liquid 19 is filled in the hollow16, the tip end portion of the conductive thin film 20 is in contactwith the medicinal liquid 19.

The pulsed power supply 4 is for applying the pulse voltage to themedicinal liquid 19 and the cell 2. This pulsed power supply 4 isconnected to the conductive thin film 20 of the cantilever 9 and theconductive thin film 12 of the scanning stage 8, respectively, andapplies the pulse voltage between the conductive thin film 20 and theconductive thin film 12 as shown in FIGS. 1 and 2. Here, since themedicinal liquid 19 has conductivity and a cell membrane 22 and cellfluid 23 forming the cell 2 also have conductivity, a current passesbetween the conductive thin film 20 and the conductive thin film 12. Atthis time, the binding force acting between the respective particlesforming the medicinal liquid 19 is cut off due to reception of anelectric shock and the medicinal liquid 19 that has lost cohesion flowsoutside the probe 17 through the fine hole 21. The amount of outflow ofthe medicinal liquid 19 can be adjusted by controlling magnitude of thepulse voltage. Moreover, since the current also flows through the cell2, a fine breakdown hole 24 is formed in the cell membrane 22 due to atransitory dielectric breakdown and the medicinal liquid 19electrophoresed by the pulse voltage passes through the breakdown hole24 in the cell membrane 22 and is taken into the cell 2. Since thebreakdown hole 24 formed in the cell membrane 22 is extremely fine, thehole 24 closes up soon due to the self-repairing function of the cell 2and the cell 2 does not suffer much damage. In this way, the medicinalliquid 19 filled in the hollow 16 is injected into the cell 2. Althoughthe medicinal liquid 19 flowing out through the fine hole 21 is injectedinto the cell 2 in the embodiment, the embodiment is not limited tothis. It is also possible to drop the flowing-out medicinal liquid 19 onthe cell 2. In this case, though it is not shown in detail in thedrawing, if a pair of electrodes is provided in contact with themedicinal liquid 19 and a pulse voltage is applied between therespective electrodes, a dielectric breakdown does not occur in the cell2 and the flowing-out medicinal liquid 19 is dropped on the cell 2without being taken into the cell 2. As a means for cutting off thebinding force acting between the respective particles of the medicinalliquid 19, besides application of the electric shock to the medicinalliquid 19 as in the embodiment, for example, a method in which amechanical shock is applied by a pulse laser may be used.

The control portion 5 controls the operations of the AFM 3 and thepulsed power supply 4. The control portion 5 causes the scanning stage 8to scan in horizontal directions, i.e., the directions of the X and Yaxes while controlling the scan in the direction of the Z axis, i.e. avertical direction so that the atomic force acting between the probe 17of the cantilever 9 and the cell 2 becomes constant. At this time, afeedback amount in the direction of the Z axis corresponding to aposition in the directions of the X and Y axes of the scanning stage 8is detected as an output voltage of the control portion 5 and is outputas a three-dimensional image on a screen through an arithmetical unit(not shown) in the computer 6 to thereby measure the surface profile ofthe cell 2 highly precisely. In the embodiment, the scanning stage 8 ismoved to scan the surface of the cell 2 while fixing the position of thecantilever 9. On the contrary, it is also possible to move thecantilever 9 to scan the surface of the cell 2 while fixing the positionof the scanning stage 8. In this case, the control portion 5 may controlthe operation of the cantilever 9. The control portion 5 also controlsthe operation of the pulsed power supply 4. While causing the scanningstage 8 to scan to observe the surface of the cell 2, it is possible toapply the voltage pulse to inject or drop the medicinal liquid 19 intoor on the cell 2 when the cantilever 9 has reached a desired position.Although the case where one cell 2 is placed on the scanning stage 8 isdescribed as an example in the embodiment, it is also possible toarrange a plurality of cells 2 on the scanning stage 8 and to inject ordrop the medicinal liquid 19 only into or on an arbitrary cell 2selected from the cells 2. After injection of the medicinal liquid 19into the cell 2 in this manner, it is possible to observe the surfaceprofile of the cell 2 by using the AFM3 to check if the medicinal liquid19 has been injected into the cell 2 reliably and a degree of breakageof the cell 2 due to application of the pulse voltage, which allows moreaccurate cell manipulation.

A procedure of the cell manipulation by using the biosample manipulationapparatus 1 will be described below. First, predetermined medicinalliquid 19 to be injected into or dropped on the cell 2 is filled in thehollow 16 in the cantilever 9. Next, the cell 2 to be manipulated isplaced on the scanning stage 8 and positioning is carried out to bringthe probe 17 of the cantilever 9 right above the cell 2 while observingthrough the optical microscope 7. Then, after the scanning stage 8 iscaused to scan in the direction of the Z axis to bring the probe 17close to the cell 2, the scanning stage 8 is caused to scan in thedirections of the X and Y axes. When the probe 17 has reached a desiredposition, the pulsed power supply 4 is operated to apply a pulse voltageof predetermined magnitude between the respective conductive thin films12 and 20. As a result, the medicinal liquid 19 flows out of the finehole 21 of the cantilever 9 and is injected into or dropped on the cell2. Then, change in the surface profile of the cell 2 is observed throughthe AFM3 and change occurring in the cell 2, e.g., a manner ofexpression of the gene injected into the cell 2 is observed through theoptical microscope 7.

Next, a second embodiment of the invention will be described withreference to the drawing. A biosample manipulation apparatus 30according to the present embodiment is characterized in that it isdifferent in a structure of a cantilever 31 from the biosamplemanipulation apparatus 1 in the first embodiment. Other structures arethe same as those in the first embodiment and detailed description ofthem will be omitted here. FIG. 4 is a schematic perspective view of thestructure of the cantilever 31 according to the embodiment. In FIG. 4,the same components as those in FIG. 3 are provided with the samereference numerals. As shown in FIG. 4, the cantilever 31 includes abeam portion 15 with its base end supported in a cantilever manner by aholder 14, a probe 17 formed to protrude from a tip end of the beamportion 15 and having a hollow 16, a mask 18 applied to an inner wallface of the probe 17, medicinal liquid 19 filled in the hollow 16, aconductive thin film 20 provided to extend from a surface of the beamportion 15 to the inner wall face of the probe 17, and a fine hole 32connecting the hollow 16 and an outside of the probe 17.

In the cantilever 9, the fine hole 32 is not positioned on a lineconnecting a vertex of the hollow 16 and a vertex of the probe 17 andformed in a position slightly displaced from the line. As a result, thevertex of the probe 17 is not chipped and remains as a probe point 33 ina sharp-pointed shape. This probe point 33 is formed to have a radius ofcurvature of about 10 nm and therefore can scan the surface of the cell2 at higher resolution as compared with the cantilever 9 in which thetip end of the probe 17 is formed of the fine hole 21 having the openingdiameter of about 20 to 500 nm. Here, in order to allow the medicinalliquid 19 to flow out to a predetermined position designated by theprobe point 33 more accurately, it is preferable to form the fine hole32 in a closer position to the probe point 33.

Next, a third embodiment of the invention will be described withreference to the drawing. A biosample manipulation apparatus 40according to the present embodiment is characterized in that it isdifferent in a structure of a cantilever 41 from the biosamplemanipulation apparatus 1 in the first embodiment. Other structures arethe same as those in the first embodiment and detailed description ofthem will be omitted here. FIG. 5 is a schematic perspective view of thestructure of the cantilever 41 according to the embodiment. In FIG. 5,the same components as those in FIG. 3 are provided with the samereference numerals. As shown in FIG. 5, the cantilever 41 includes abeam portion 15 with its base end supported in a cantilever manner by aholder 14, a probe 17 formed to protrude from a tip end of the beamportion 15 and having a hollow 16, a mask 18 applied to an inner wallface of the probe 17, medicinal liquid 19 filled in the hollow 16, aconductive thin film 20 provided to extend from a surface of the beamportion 15 to the inner wall face of the probe 17, a fine hole 21connecting the hollow 16 and an outside of the probe 17, and aprotruding portion 42 provided to a tip end portion of the probe 17 andfunctioning as a probe point.

In the cantilever 41, the fine hole 21 is formed from a vertex of thehollow 16 toward a vertex of the probe 17 and the vertex of the probe 17is chipped similarly to the cantilever 9 in the first embodiment. On theother hand, the protruding portion 42 is formed in a triangular shapehaving a sharp-pointed corner portion 43 and one end edge of theprotruding portion 42 is secured to an outer wall face of the probe 17so that the sharp-pointed corner portion 43 is positioned below the finehole 21. By bringing the sharp-pointed corner portion 43 of theprotruding portion 42 extremely close to the surface of the cell 2, itis possible to scan the surface of the cell 2 at higher resolution ascompared with the cantilever 9 in the first embodiment. It is essentialonly that the protruding portion 42 have the sharp-pointed cornerportion 43 and the shape of the protruding portion 42 is not limited tothe triangle but may be changed properly in design.

Next, a fourth embodiment of the invention will be described withreference to the drawing. A biosample manipulation apparatus 50according to the present embodiment is characterized in that it isdifferent in a structure of a cantilever 51 from the biosamplemanipulation apparatus 1 in the first embodiment. Other structures arethe same as those in the first embodiment and detailed description ofthem will be omitted here. FIG. 6 is a schematic perspective view of thestructure of the cantilever 51 according to the embodiment. In FIG. 6,the same components as those in FIG. 3 are provided with the samereference numerals. As shown in FIG. 6, the cantilever 51 includes abeam portion 15 with its base end supported in a cantilever manner by aholder 14, a probe 17 formed to protrude from a tip end of the beamportion 15 and having a hollow 16, a mask 18 applied to an inner wallface of the probe 17, medicinal liquid 19 filled in the hollow 16, aconductive thin film 20 provided to extend from a surface of the beamportion 15 to the inner wall face of the probe 17, a fine hole 21connecting the hollow 16 and an outside of the probe 17, and a nanotube52 attached to a tip end portion of the probe 17.

In the cantilever 51, the fine hole 21 is formed from a vertex of thehollow 16 toward a vertex of the probe 17 and the vertex of the probe 17is chipped similarly to the cantilever 9 in the first embodiment. On theother hand, the nanotube 52 is made of carbon or the like and a base endportion of the nanotube 52 is secured to an outer wall face of the probe17 so that a tip end portion of the nanotube 52 protrudes below the tipend of the probe 17. An opening diameter of the nanotube 52 is extremelyminute. By bringing the tip end of the nanotube 52 extremely close tothe surface of the cell 2, it is possible to scan the surface of thecell 2 at higher resolution as compared with the cantilever 9 in thefirst embodiment.

Next, a fifth embodiment of the invention will be described withreference to the drawing. A biosample manipulation apparatus 60according to the present embodiment is characterized in that it isdifferent in a structure of a cantilever 61 from the biosamplemanipulation apparatus 1 in the first embodiment. Other structures arethe same as those in the first embodiment and detailed description ofthem will be omitted here. FIG. 7 is a schematic perspective view of thestructure of the cantilever 61 according to the embodiment. In FIG. 7,the same components as those in FIG. 3 are provided with the samereference numerals. As shown in FIG. 7, the cantilever 61 includes abeam portion 15 with its base end supported in a cantilever manner by aholder 14, a probe 17 formed to protrude from a tip end of the beamportion 15 and having a hollow 16, a mask 18 applied to an inner wallface of the probe 17, reactive gas (fluid) 62 filled in a tank (notshown), a feed nozzle 63 for feeding the reactive gas 62 to thecantilever 61, a conductive thin film 20 provided to extend from asurface of the beam portion 15 to the inner wall face of the probe 17,and a fine hole 21 connecting the hollow 16 and an outside of the probe17.

The reactive gas 62 is a generic term used to refer to substances whichdo not vaporize in a vacuum and which excite various chemical reactionswith a surface of a substance and includes compounds, mixtures, and thelike as well as elements. As the reactive gas 62, it is possible to usehalogen gas represented by HF or HCl, gas cyanide represented by C4H5Nor CH3CH2CN, and the like, for example. Besides those, it is possible touse one in a solid or liquid state at room temperature after vaporizingit by heating or the like. Although it is not shown in detail in thedrawing, the reactive gas 62 is filled in the tank that is a vacuumchamber. On the other hand, one end of the feed nozzle 63 is connectedto the tank and the other end of the nozzle 63 is throttled thin andinserted into the hollow 16. The reactive gas 62 shot from the feednozzle 63 is not shot outside the probe 17 through the fine hole 21 butstays in the hollow 16 since the opening diameter of the fine hole 21 isextremely minute and respective particles forming the reactive gas 62are bound together by action of atomic force or the like similarly tothe above-described medicinal liquid 19. By application of the pulsevoltage to the reactive gas 62 from the conductive thin film 20, thebinding force acting between the respective particles is cut off and thereactive gas 62 flows outside the probe 17. In this way, by allowing thereactive gas 62 to flow out of the fine hole 21 and adhere to thesurface of the cell 2, various chemical reactions are excited betweenthe cell 2 and the reactive gas 62 and this change is observed by theAFM3 and the optical microscope 7.

INDUSTRIAL APPLICABILITY

In the invention, not only the cantilever provided to the atomic forcemicroscope but also cantilevers provided to other scanning probemicroscopes may be used.

1. A biosample manipulation method for injecting a physiologicallyactive substance or a substance for exciting a chemical reaction into abiosample comprising the steps of: providing a scanning probe microscopewith a cantilever comprising a beam portion with its base end supportedin a cantilever manner, a probe formed to protrude from a tip end of thebeam portion and having a hollow in itself, a fine hole formed topenetrate from the hollow to an outside of the probe through the probe,and a first electrode provided in the hollow; filling fluid includingthe physiologically active substance or the substance for exciting thechemical reaction and having conductivity in the hollow in thecantilever; placing the biosample on a second electrode formed of aconductive thin film; bringing the probe of the cantilever close to thebiosample; applying a pulse voltage between the first electrode and thesecond electrode the thereby allow the fluid to flow out of the finehole and inject the fluid into the biosample; and observing change in asurface profile of the biosample through the scanning probe microscope.2. A biosample manipulation method for dropping a physiologically activesubstance or a substance for exciting a chemical reaction on a biosamplecomprising the steps of: providing the scanning probe microscope withthe cantilever comprising a beam portion with its base end supported ina cantilever manner, the probe formed to protrude from the beam portionand having the hollow in itself, the fine hole formed to penetrate fromthe hollow to an outside of the probe through the probe, and a firstelectrode and a second electrode provided in the hollow; filling fluidincluding the physiologically active substance or the substance forexciting the chemical reaction and having conductivity in the hollow inthe cantilever; bringing the probe of the cantilever close to thebiosample; applying a pulse voltage between the first electrode and thesecond electrode to thereby allow the fluid to flow out of the fine holeand drop the fluid on the biosample; and observing change in a surfaceprofile of the biosample through the scanning probe microscope.
 3. Thebiosample manipulation method according to claim 1 and furthercomprising the step of observing manipulation of the biosample and innerchange of the biosample after the manipulation through an opticalmicroscope.
 4. The biosample manipulation apparatus according to claim3, wherein the fluid outflow means comprises a pair of electrodesprovided in contact with the fluid and a pulsed power supply forapplying a pulse voltage between the respective electrodes.
 5. Thebiosample manipulation apparatus according to claim 1, wherein the beamportion, the probe, and the fine hole constitute a probe of a scanningprobe microscope.
 6. The biosample manipulation apparatus according toclaim 1 and further comprising a manipulation observing means forobserving manipulation of the biosample and change of the biosampleafter the manipulation.
 7. The biosmaple manipulation method accordingto claim 2 and further comprising the step of observing manipulation ofthe biosample and inner change of the biosample after the manipulationthrough an optical microscope.
 8. The biosample manipulation apparatusaccording to claim 7, wherein the fluid outflow means comprises a pairof electrodes provided in contact with the fluid and a pulsed powersupply for applying a pulse voltage between the respective electrodes.9. The biosample manipulation apparatus according to claim 2, whereinthe beam portion, the probe, and the fine hole constitute a probe of ascanning probe microscope.
 10. The biosample manipulation apparatusaccording to claim 3, wherein the beam portion, the probe, and the finehole constitute a probe of a scanning probe microscope.
 11. Thebiosample manipulation apparatus according to claim 4, wherein the beamportion, the probe, and the fine hole constitute a probe of a scanningprobe microscope.
 12. The biosample manipulation apparatus according toclaim 7, wherein the beam portion, the probe, and the fine holeconstitute a probe of a scanning probe microscope.
 13. The biosamplemanipulation apparatus according to claim 8, wherein the beam portion,the probe, and the fine hole constitute a probe of a scanning probemicroscope.
 14. The biosample manipulation apparatus according to claim2 and further comprising a manipulation observing means for observingmanipulation of the biosample and change of the biosample after themanipulation.
 15. The biosample manipulation apparatus according toclaim 3 and further comprising a manipulation observing means forobserving manipulation of the biosample and change of the biosampleafter the manipulation.
 16. The biosample manipulation apparatusaccording to claim 4 and further comprising a manipulation observingmeans for observing manipulation of the biosample and change of thebiosample after the manipulation.
 17. The biosample manipulationapparatus according to claim 5 and further comprising a manipulationobserving means for observing manipulation of the biosample and changeof the biosample after the manipulation.
 18. The biosample manipulationapparatus according to claim 7 and further comprising a manipulationobserving means for observing manipulation of the biosample and changeof the biosample after the manipulation.
 19. The biosample manipulationapparatus according to claim 8 and further comprising a manipulationobserving means for observing manipulation of the biosample and changeof the biosample after the manipulation.